Abstract: Gene editing in immune cells, particularly T cells and natural killer (NK) cells, holds tremendous potential for developing targeted immunotherapies and understanding immune cell biology. This review focuses on transfection reagents and techniques utilized for introducing gene-editing tools, such as CRISPR/Cas9, into immune cells. We discuss the challenges associated with immune cell transfection, explore different transfection methods and reagents, and highlight recent advancements and applications in immune cell gene editing. This comprehensive review provides valuable insights for researchers and clinicians aiming to harness the power of gene editing in immune cells.

Introduction: Gene editing has revolutionized biomedical research, and its application in immune cells has opened new avenues for targeted immunotherapies and the exploration of immune cell functions. Efficient delivery of gene-editing tools into immune cells, such as T cells and NK cells, is crucial for successful genome engineering. This review examines various transfection reagents and techniques employed for introducing gene-editing tools, with a focus on CRISPR/Cas9, into immune cells.

Challenges in Immune Cell Transfection:

1. Primary Cell Nature: Immune cells, particularly primary T cells and NK cells, have unique characteristics that pose challenges for efficient transfection, including limited cell numbers and resistance to exogenous material uptake.

Transfection Techniques for Immune Cell Gene Editing:

1. Viral Transduction:
   • Lentiviral Vectors: Lentiviral transduction is widely used for gene editing in immune cells, providing stable and long-term expression of gene-editing tools.
   • Adenoviral Vectors: Adenoviral vectors efficiently transduce immune cells but result in transient gene expression, making them suitable for short-term experiments.
2. Electroporation:
   • Nucleofection: Electroporation-based methods, such as nucleofection, enable efficient gene delivery into immune cells by creating transient pores in the cell membrane.
3. Lipid-based Transfection Reagents:
   • Lipofection: Lipid-based transfection reagents facilitate the delivery of gene-editing tools into immune cells by forming complexes with nucleic acids, promoting cellular uptake and intracellular release.
4. Physical Methods:
   • Particle-Mediated Transfection: Techniques like biolistic particle delivery (gene gun) propel nucleic acid-coated particles into immune cells, allowing gene delivery.

Recent Advancements and Applications:

1. CRISPR/Cas9-Mediated Gene Editing:
   • Targeted Gene Knockout: CRISPR/Cas9 enables precise disruption of genes in immune cells, aiding in functional studies and immunotherapeutic applications.
   • Gene Insertion and Correction: CRISPR/Cas9 facilitates the insertion or correction of genes in immune cells for therapeutic purposes or generating modified cell therapies.
2. Gene Editing in CAR-T Cells:
   • Introduction of Chimeric Antigen Receptors (CARs): Gene editing techniques combined with transfection methods enable efficient CAR-T cell engineering, enhancing their antigen specificity and therapeutic potential.
3. Gene Editing in NK Cells:
   • Enhancing NK Cell Function: Gene editing allows the manipulation of NK cell receptors or signaling pathways to enhance their cytotoxicity and tumor-targeting abilities.

Conclusion: Efficient transfection of immune cells, particularly T cells and NK cells, is vital for successful gene editing and the development of targeted immunotherapies. Various transfection reagents and techniques, including viral transduction, electroporation, lipid-based reagents, and physical methods, have been employed for introducing gene-editing tools into immune cells. Continued advancements in transfection methods and reagents will further improve the efficiency and specificity of immune cell gene editing, opening new possibilities for personalized immunotherapies and advancing our understanding of immune cell biology.